Human coronavirus OC43 nanobody neutralizes virus and protects mice from infection.

Human coronavirus (hCoV) OC43 is endemic to global populations and usually causes asymptomatic or mild upper respiratory tract illness. Here, we demonstrate the neutralization efficacy of isolated nanobodies from alpacas immunized with the S1B and S1C domain of the hCoV-OC43 spike glycoprotein. A total of 40 nanobodies bound to recombinant OC43 protein with affinities ranging from 1 to 149 nM. Two nanobodies WNb 293 and WNb 294 neutralized virus at 0.21 and 1.79 nM, respectively. Intranasal and intraperitoneal delivery of WNb 293 fused to an Fc domain significantly reduced nasal viral load in a mouse model of hCoV-OC43 infection. Using X-ray crystallography, we observed that WNb 293 bound to an epitope on the OC43 S1B domain, distal from the sialoglycan-binding site involved in host cell entry. This result suggests that neutralization mechanism of this nanobody does not involve disruption of glycan binding. Our work provides characterization of nanobodies against hCoV-OC43 that blocks virus entry and reduces viral loads in vivo and may contribute to future nanobody-based therapies for hCoV-OC43 infections. IMPORTANCE: The pandemic potential presented by coronaviruses has been demonstrated by the ongoing COVID-19 pandemic and previous epidemics caused by severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Outside of these major pathogenic coronaviruses, there are four endemic coronaviruses that infect humans: hCoV-OC43, hCoV-229E, hCoV-HKU1, and hCoV-NL63. We identified a collection of nanobodies against human coronavirus OC43 (hCoV-OC43) and found that two high-affinity nanobodies potently neutralized hCoV-OC43 at low nanomolar concentrations. Prophylactic administration of one neutralizing nanobody reduced viral loads in mice infected with hCoV-OC43, showing the potential for nanobody-based therapies for hCoV-OC43 infections.

Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice.

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity.

The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.

Antibody Fc-binding profiles and ACE2 affinity to SARS-CoV-2 RBD variants.

Emerging SARS-CoV-2 variants, notably Omicron, continue to remain a formidable challenge to worldwide public health. The SARS-CoV-2 receptor-binding domain (RBD) is a hotspot for mutations, reflecting its critical role at the ACE2 interface during viral entry. Here, we comprehensively investigated the impact of RBD mutations, including 5 variants of concern (VOC) or interest-including Omicron (BA.2)-and 33 common point mutations, both on IgG recognition and ACE2-binding inhibition, as well as FcγRIIa- and FcγRIIIa-binding antibodies, in plasma from two-dose BNT162b2-vaccine recipients and mild-COVID-19 convalescent subjects obtained during the first wave using a custom-designed bead-based 39-plex array. IgG-recognition and FcγR-binding antibodies were decreased against the RBD of Beta and Omicron, as well as point mutation G446S, found in several Omicron sub-variants as compared to wild type. Notably, while there was a profound decrease in ACE2 inhibition against Omicron, FcγR-binding antibodies were less affected, suggesting that Fc functional antibody responses may be better retained against the RBD of Omicron in comparison to neutralization. Furthermore, while measurement of RBD-ACE2-binding affinity via biolayer interferometry showed that all VOC RBDs have enhanced affinity to human ACE2, we demonstrate that human ACE2 polymorphisms, E35K (rs1348114695) has reduced affinity to VOCs, while K26R (rs4646116) and S19P (rs73635825) have increased binding kinetics to the RBD of VOCs, potentially affecting virus-host interaction and, thereby, host susceptibility. Collectively, our findings provide in-depth coverage of the impact of RBD mutations on key facets of host-virus interactions.

Nanobodies against Pfs230 block Plasmodium falciparum transmission.

Transmission blocking interventions can stop malaria parasite transmission from mosquito to human by inhibiting parasite infection in mosquitos. One of the most advanced candidates for a malaria transmission blocking vaccine is Pfs230. Pfs230 is the largest member of the 6-cysteine protein family with 14 consecutive 6-cysteine domains and is expressed on the surface of gametocytes and gametes. Here, we present the crystal structure of the first two 6-cysteine domains of Pfs230. We identified high affinity Pfs230-specific nanobodies that recognized gametocytes and bind to distinct sites on Pfs230, which were isolated from immunized alpacas. Using two non-overlapping Pfs230 nanobodies, we show that these nanobodies significantly blocked P. falciparum transmission and reduced the formation of exflagellation centers. Crystal structures of the transmission blocking nanobodies with the first 6-cysteine domain of Pfs230 confirm that they bind to different epitopes. In addition, these nanobodies bind to Pfs230 in the absence of the prodomain, in contrast with the binding of known Pfs230 transmission blocking antibodies. These results provide additional structural insight into Pfs230 domains and elucidate a mechanism of action of transmission blocking Pfs230 nanobodies.

Mutational and biophysical robustness in a prestabilized monobody.

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

A TCR ß-Chain Motif Biases toward Recognition of Human CD1 Proteins.

High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR ß-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

TCR bias and affinity define two compartments of the CD1b-glycolipid-specific T Cell repertoire.

Current views emphasize TCR diversity as a key feature that differentiates the group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 systems. Whereas TCR sequence motifs define CD1d-reactive NKT cells, the available data do not allow a TCR-based organization of the group 1 CD1 repertoire. The observed TCR diversity might result from donor-to-donor differences in TCR repertoire, as seen for MHC-restricted T cells. Alternatively, diversity might result from differing CD1 isoforms, Ags, and methods used to identify TCRs. Using CD1b tetramers to isolate clones recognizing the same glycolipid, we identified a previously unknown pattern of V gene usage (TRAV17, TRBV4-1) among unrelated human subjects. These TCRs are distinct from those present on NKT cells and germline-encoded mycolyl lipid-reactive T cells. Instead, they resemble the TCR of LDN5, one of the first known CD1b-reactive clones that was previously thought to illustrate the diversity of the TCR repertoire. Interdonor TCR conservation was observed in vitro and ex vivo, identifying LDN5-like T cells as a distinct T cell type. These data support TCR-based organization of the CD1b repertoire, which consists of at least two compartments that differ in TCR sequence motifs, affinity, and coreceptor expression.

User perspectives of digital manufacturing for lower-limb prosthetic sockets.

BACKGROUND: There is growing interest to use digital technology (DT) for manufacturing lower-limb prosthetic sockets to improve efficiency and clinical outcomes. However, little is known about how lower-limb prosthesis users perceive DTs, such as 3D scanning and 3D printing. OBJECTIVES: This study aimed to provide an understanding of perceptions and experiences with DT for prosthetic socket manufacturing from the perspective of prosthesis users. STUDY DESIGN: A qualitative descriptive research study. METHODS: Nine lower-limb prosthesis users (mean age 56; 5 female; 4 male) participated in one-on-one semistructured telephone interviews. Inductive thematic analysis was performed to identify a codebook and emerging themes from the interview transcripts. RESULTS: Two major themes were identified: (1) expectations and prioritization of 3D printed socket usability and (2) facilitators and barriers to uptake of DT among patients. CONCLUSION: DT methods were found to be acceptable and feasible from a patient perspective, although technological advancements are still required, and real-time communication about the process may be vital for ensuring patient engagement. Consideration of these findings may improve patient satisfaction to emerging prosthesis treatment plans and ultimately support widespread adoption of DT as an additional tool for fabricating prosthetic sockets.

An enhanced Runge Kutta boosted machine learning framework for medical diagnosis.

With the development and maturity of machine learning methods, medical diagnosis aided with machine learning methods has become a popular method to assist doctors in diagnosing and treating patients. However, machine learning methods are greatly affected by their hyperparameters, for instance, the kernel parameter in kernel extreme learning machine (KELM) and the learning rate in residual neural networks (ResNet). If the hyperparameters are appropriately set, the performance of the classifier can be significantly improved. To boost the performance of the machine learning methods, this paper proposes to improve the Runge Kutta optimizer (RUN) to adaptively adjust the hyperparameters of the machine learning methods for medical diagnosis purposes. Although RUN has a solid mathematical theoretical foundation, there are still some performance defects when dealing with complex optimization problems. To remedy these defects, this paper proposes a new enhanced RUN method with a grey wolf mechanism and an orthogonal learning mechanism called GORUN. The superior performance of the GORUN was validated against other well-established optimizers on IEEE CEC 2017 benchmark functions. Then, the proposed GORUN is employed to optimize the machine learning models, including the KELM and ResNet, to construct robust models for medical diagnosis. The performance of the proposed machine learning framework was validated on several medical data sets, and the experimental results have demonstrated its superiority.

A qualitative study exploring healthcare professionals' perceptions of lower limb 3D printed sockets.

PURPOSE: The purpose of this study was to explore healthcare professionals' (HCPs) perceptions and experiences related to 3D scanning and 3D printing for fabricating lower limb prosthetic sockets. MATERIALS AND METHODS: This study used a qualitative descriptive approach. Participants were recruited through HCPs' professional associations, social media posts, and snowball sampling. Purposive sampling was used to attain variation in provider type. One-on-one telephone interviews were conducted using a semi-structured interview guide. Inductive thematic analysis was performed to identify the main themes. RESULTS: Three themes were identified: (1) 3D scanning of the residual limb for designing prosthetic sockets is perceived as clean, quick, and convenient; (2) concerns about the strength and safety of 3D printed sockets for long-term use; (3) Adoption of 3D scanning and 3D printing technology for fabricating prosthetic sockets. CONCLUSION: We identified perceived benefits and challenges with digital technologies for fabricating prosthetic sockets. To increase adoption, more research demonstrating its efficacy compared to conventional methods, increasing 3D printing material quality, and improving software training programs are needed.Implications for Rehabilitation3D printing and 3D scanning are emerging digital technologies that can be used as alternative methods for prosthetic socket manufacturing in the field of rehabilitation.Our research identified perceived benefits of using digital technologies for fabricating prosthetics sockets (3D scanning is perceived as clean, quick, and convenient) and perceived challenges (concerns about the strength and safety of 3D printed sockets for long-term use and a prolonged learning curve).To increase adoption of these digital technologies, more training should be provided to prosthetists and support provided to integrate new processes into staff workloads.

Correction to "Substrate Dynamics Contribute to Enzymatic Specificity in Human and Bacterial Methionine Adenosyltransferases".

[This corrects the article DOI: 10.1021/jacsau.1c00464.].

Structure of the Pf12 and Pf41 heterodimeric complex of Plasmodium falciparum 6-cysteine proteins.

During the different stages of the Plasmodium life cycle, surface-associated proteins establish key interactions with the host and play critical roles in parasite survival. The 6-cysteine (6-cys) protein family is one of the most abundant surface antigens and expressed throughout the Plasmodium falciparum life cycle. This protein family is conserved across Plasmodium species and plays critical roles in parasite transmission, evasion of the host immune response and host cell invasion. Several 6-cys proteins are present on the parasite surface as hetero-complexes but it is not known how two 6-cys proteins interact together. Here, we present a crystal structure of Pf12 bound to Pf41 at 2.85 Å resolution, two P. falciparum proteins usually found on the parasite surface of late schizonts and merozoites. Our structure revealed two critical interfaces required for complex formation with important implications on how different 6-cysteine proteins may interact with each other. Using structure-function analyses, we identified important residues for Pf12-Pf41 complex formation. In addition, we generated 16 nanobodies against Pf12 and Pf41 and showed that several Pf12-specific nanobodies inhibit Pf12-Pf41 complex formation. Using X-ray crystallography, we were able to describe the structural mechanism of an inhibitory nanobody in blocking Pf12-Pf41 complex formation. Future studies using these inhibitory nanobodies will be useful to determine the functional role of these two 6-cys proteins in malaria parasites.

Knowledge, Attitudes, and Practices of COVID-19 Vaccination among Adults in Singapore: A Cross-Sectional Study.

Public health measures promoting compliance of COVID-19 vaccination requires understanding of knowledge, attitudes, and practices (KAP). This study explored the KAP and risk factors influencing COVID-19 vaccination, including changes in preventive practices before and after vaccination in a high-income country, Singapore. An online cross-sectional study among Singaporeans and permanent residents aged 21 years and older was conducted from July to August 2021. Univariate and multivariable logistic regressions using RStudio version 1.4.1106 was performed to assess associations between demographic factors, KAP, and vaccination status. P values < 0.05 were considered statistically significant. A total of 869 respondents completed the survey. Individuals with higher knowledge (adjusted odds ratio [aOR] = 2.00, P = 0.024), perceived efficacy (aOR = 1.19, P = 0.004), perceived safety (aOR = 1.20, P = 0.005), and willingness to uptake (aOR = 1.55, P < 0.001) scores were more likely to be vaccinated. There was a significant increase in the use of proper handwashing techniques among the vaccinated group before and after vaccinations. The governmental risk communication approaches have been useful in instilling high levels of vaccine knowledge. High levels of good attitudes about and knowledge of COVID-19 vaccination were associated with a high level of vaccination practices. Good perceived vaccine efficacy and confidence in government were also associated with positive vaccine uptake. This study paves the way for more targeted government measures to be implemented to improve vaccination rates of COVID-19 booster vaccines in a high-income country like Singapore.

Substrate Dynamics Contribute to Enzymatic Specificity in Human and Bacterial Methionine Adenosyltransferases.

Protein conformational changes can facilitate the binding of noncognate substrates and underlying promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here, we analyze human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, noncognate substrates bind in a stable conformation to allow catalysis. In contrast, noncognate substrates sample stable productive binding modes less frequently in eMAT owing to altered mobility in the enzyme active site. Different cellular concentrations of substrates likely drove the evolutionary divergence of substrate specificity in these orthologues. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated levels in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the effects of substrate and enzyme dynamics.

Landscape of human antibody recognition of the SARS-CoV-2 receptor binding domain.

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.

CD4+ T cell-mediated HLA class II cross-restriction in HIV controllers.

Rare individuals, termed HIV controllers, spontaneously control HIV infection by mounting efficient T cell responses against the virus. Protective CD4+ T cell responses from HIV controllers involve high-affinity public T cell receptors (TCRs) recognizing an immunodominant capsid epitope (Gag293) presented by a remarkably broad array of human leukocyte antigen (HLA) class II molecules. Here, we determine the structures of a prototypical public TCR bound to HLA-DR1, HLA-DR11, and HLA-DR15 molecules presenting the Gag293 epitope. TCR recognition was driven by contacts with the Gag293 epitope, a feature that underpinned the extensive HLA cross-restriction. These high-affinity TCRs promoted mature immunological synapse formation and cytotoxic capacity in both CD4+ and CD8+ T cells. The public TCRs suppressed HIV replication in multiple genetic backgrounds ex vivo, emphasizing the functional advantage conferred by broad HLA class II cross-restriction.

T cell receptor recognition of CD1b presenting a mycobacterial glycolipid.

CD1 proteins present microbial lipids to T cells. Germline-encoded mycolyl lipid-reactive (GEM) T cells with conserved αß T cell receptors (TCRs) recognize CD1b presenting mycobacterial mycolates. As the molecular basis underpinning TCR recognition of CD1b remains unknown, here we determine the structure of a GEM TCR bound to CD1b presenting glucose-6-O-monomycolate (GMM). The GEM TCR docks centrally above CD1b, whereby the conserved TCR α-chain extensively contacts CD1b and GMM. Through mutagenesis and study of T cells from tuberculosis patients, we identify a consensus CD1b footprint of TCRs present among GEM T cells. Using both the TCR α- and ß-chains as tweezers to surround and grip the glucose moiety of GMM, GEM TCRs create a highly specific mechanism for recognizing this mycobacterial glycolipid.

Early intervention in planning end-of-life care with ambulatory geriatric patients: results of a pilot trial.

BACKGROUND: A large discrepancy exists between the wishes of dying patients and their actual end-of-life care. However, retrospective clinical experience suggests that early advance care planning (ACP) can markedly reduce this discrepancy. This article describes a randomized trial to evaluate the short-term clinical utility of early ACP. We also assessed the feasibility of performing a larger prospective study to document long-term outcomes. METHODS: Ambulatory geriatric patients (N = 61) were randomized to either a control group, which received only a Massachusetts Health Care Proxy form to complete, or an intervention group, in which each patient and health care agent discussed ACP with a trained nurse facilitator. The benefits and burdens of life-sustaining treatments were discussed, and patient goals and preferences for these treatments were documented. RESULTS: Two-month follow-up revealed that the intervention achieved higher congruence between agents and patients in their understanding of patients' end-of-life care preferences, with 76% (19/25) in complete agreement vs 55% (12/22) of the controls (effect size [ES] = -0.43). There was also a greater increase in patient knowledge about ACP in the intervention group (ES = 0.22). Intervention patients became less willing to undergo life-sustaining treatments for a new serious medical problem (ES = -0.25), more willing to undergo such treatments for an incurable progressive disease (ES = 0.24), and less willing to tolerate poor health states (ES = -0.78). Practical insights were gained about how to conduct a larger study more effectively. CONCLUSION: A facilitated discussion about end-of-life care between patients and their health care agents helps define and document the patient's wishes for both patient and agent.

Screening for dementia in "real world" settings: the cognitive assessment screening test: CAST.

Among elderly people who do not present with complaints of memory impairment, dementia is often missed by physicians, and time-consuming screening tests requiring expertise to administer and interpret are rarely done. Easily administered, reliable and cost effective dementia screening tests are needed for elderly individuals. The "pencil and paper" Cognitive Assessment Screening Test (CAST) takes minimal examiner time/training, and is both sensitive and specific in discriminating demented patients from healthy controls. The objectives of this study were to: (1) confirm the validity of the CAST in identifying individuals with dementia in a real-world setting (nonassisted living retirement community); (2) compare the sensitivity and specificity with other screening tests and extensive psychometric tests; and (3) assess the reliability of the CAST in test-retest conditions over time. The CAST was both sensitive and specific and showed reliability on retesting. The CAST is both simpler to administer and more accurate than other screening tests for elderly subjects.